K acetrm

FjoAcXE, CjGlcA67A, AxyAgu115A, and E-AXEAO were tested alone and as pairs of carbohydrate esterase and α-glucuronidase activities. Reactions comprised 1% (w/v) Ac-XOS in 50 mM HEPES (pH 7.0), which is within one pH unit from the optimum pH of each enzyme. Reactions were initiated by adding 0.5 µg of each enzyme; the final reaction volume was 30 µL. Reactions continued for 20 min at 30 °C and were stopped by boiling for 10 min. The samples were quickly centrifuged and release of acetic acid and d-glucuronic acid were measured using Acetic Acid (K-ACETRM, Megazyme) and d-glucuronic acid/d-galacturonic acid (K-URONIC, Megazyme) assay kits, respectively. Reaction mixtures without enzymes were used as blanks.

The activities of studied AcXEs on acetylated xylooligosaccharides were tested using highly branched acetylated and feruloylated CF-XOS (described in Appeldoorn et al., 2013 [30 (link)]) and a per-acetylated xylo-oligosaccharide mixture (DP 4–7). Acetic acid release was determined using the acetic acid kit (K-ACETRM, Megazyme). Reactions (50 µL final volume) containing CF-XOS comprised 1 µg enzyme, and 50 mM HEPES buffer (pH 7.0) and 0.5 µg of CF-XOS, and were incubated at 30 °C for 20 min and 4 h. Reactions (60 µL final volume) containing the per-acetylated xylo-oligosaccharide mixture comprised 2 µg enzyme, 50 mM HEPES buffer (pH 7.0) and 0.5 µg of per-acetylated xylo-oligosaccharide mixture, and were incubated at 30 °C for 20 min, 4 h and 20 h. The maximal acetic acid release from each substrate was determined using a modified method described by Teleman et al., 2002 [4 (link)]. Briefly, 1% (w/v) substrate was suspended in 50 µL 0.5 M NaOH for 30 min at 70 °C and 450 rpm. The reaction solutions were then neutralized, and the release of acetic acid was measured with acetic acid kit. Control reactions without enzyme were run in parallel and the amount of non-enzymatically released acetic acid was subtracted from the values of enzyme–substrate reaction mixtures.

The blood plasma samples were analyzed through an animal diagnostic service (Animal Health Laboratory, University of Guelph, Guelph, Canada). Briefly, an automated analyzer (Cobas^® c311/501 analyzer, Roche Diagnostics GmbH, Indianapolis, USA) [26 (link)] was used to measure the following blood plasma metabolic parameters: albumin (g/l), aspartate aminotransferase (AST; U/l), γ-glutamyl transpeptidase (GGT; U/l), urea (mmol/l), cholesterol (mmol/l), creatinine (mmol/l), alkaline phosphatase (ALP; U/l), creatine kinase (U/l) and lipase (U/l). Carbon dioxide (CO₂; mmol/l) levels were determined using an automated analyzer (Cobas^® 4000 c311, Roche Diagnostics GmbH, Mannheim, Germany). The determination of β-hydroxybutyrate (µmol/l) was completed using a commercial kit (Randox^®, RANDOX Laboratories Ltd., Crumlin, UK). Blood plasma acetate (g/l) was determined via spectrophotometry using a commercial kit (K-ACETRM, Megazyme© International, Wicklow, Ireland) according to the manufacturers’ protocol. A colorimetric-based assay was used to determine the concentration of total bile acids (BA; µmol/l) (Diazyme total bile acids, Diazyme©, Poway, USA).